Our efforts to elucidate the structure-function relationships associated with the cholera toxin family of enterotoxins have continued. These toxins, which act by ADP-ribosylating eukaryotic G proteins, are considered to be important antigens for potential inclusion into vaccines for diarrheal disease mediated by organisms such as Vibrio cholerae and enterotoxigenic Escherichia coli. These toxins also possess potent mucosal adjuvant properties. Accordingly, we are attempting to identify the primary structural requirements associated with toxicity, antigenicity/immunogenicity, and adjuvanticity. We have successfully expressed the genes for both the entire operon and the gene for the enzymatically active A subunit of E. coli heat-labile enterotoxin (LT) in vectors that permit single stranded mutagenesis procedures. Site-specific mutants of codons encoding various amino acids have allowed us to identify residues in the A subunit that appear to be critical to the maintenance of enzymatic activity. Such residues include glutamic acid 110, glutamic acid 112, tryptophan 124, and histidine 44. Mutations within the amino-terminal region of the A subunit also reduce enzymatic activity, but impart gross conformational changes and as such do not appear to be suitable targets for the production of inactivated vaccine candidates. More recently, similar mutations have been introduced into the operon encoding the entire toxin to verify that elimination of enzymatic activity also abrogates toxicity. In addition, we have determined, using site-directed mutagenesis that trypsin-like proteolytic cleavage of LT is not requisite to toxicity, a fact that alters our perception of the intracellular processing and fate of the toxin.